Non-autonomous cell redox-pairs dictate niche homeostasis in multi-lineage stem populations

Reviewer #1 (Public Review):

The manuscript by Majhi and colleagues describes the effects of manipulating ROS levels in somatic stem cells of the testis on overall testis architecture, signaling, and function. The conclusions made by the authors are somewhat difficult to judge as the changes to the testis cell types are mostly not apparent in the representative images shown. This is true in examining gstD1-GFP expression and in the analysis of cell types and behaviours (e.g. cell cycle) and cell signaling pathway activity. Thus, the reader is left to try and interpret the quantification of the data to justify the authors' conclusions, but it is often not clear how the quantification was accomplished. For example, it is not clear how CySC vs. GSC quantification is done when the molecular markers used do not define the surface of these cells (plasma membrane) and mark different cellular compartments (Tj is nuclear while Vasa is perinuclear or cytoplasmic). Why the changes reported in quantification are not apparent in the specific example images chosen for the figures is worrisome. I'm much more used to being able to clearly see what the authors are reporting in the images, and then using the quantification to illustrate the range of data observed and demonstrate statistical significance. For this reason, I'm very concerned about the strength and validity of the conclusions. In addition, while many different characteristics of the testis somatic and germline cells are analyzed, a general and consistent view of how ROS affects these cells is not presented. In particular, one of the principle conclusions, that ROS signaling in the CySCs affects ROS signaling in the GSCs, is not well-supported by the data presented.

Specific Comments:

In Figure 1, it is very difficult to identify where CySCs end and GSCs begin without using a cell surface marker for these different cell types. In addition, the methods for quantifying the mitochondrial distribution in GSCs vs. CySCs are very much unclear, and appear to rely on colocalization with molecular markers that are not in the same cellular compartment (Tj-nuclear vs Vasa-perinuclear and cytoplasmic), the reader has no way to determine the validity of the mitochondrial distribution. Similarly, the labeling with gstD1-GFP is also very much unclear - I see little to no GFP signal in either GSCs or CySCs in panels 1G-K. Lastly, while the expression of SOD in CySCs does increase the gstD1-GFP signal in CySCs, the effects on GSCs claimed by the authors are not apparent.

In Figure 2, while the cell composition of the niche region does appear to be different from controls when SOD1 is knocked down in the CySCs, at least in the example images shown in Figures 2A and B, how cell type is quantified in Figures 2E-G is very much unclear in the figure and methods. Are these counts of cells contacting the niche? If so, how was that defined? Or were additional regions away from the niche also counted and, if so, how were these regions defined?

In Figure 3, it is quite interesting that there is an increase in Eya+, differentiating cyst cells in SOD1 knockdown animals, and that these Eya+ cells appear closer to the niche than in controls. However, this seems at odds with the proliferation data presented in Figure 2, since Eya+ somatic cells do not normally divide at all. Are they suggesting that now differentiating cyst cells are proliferative? In addition, it is important for them to show example images of the changes in Socs36E and ptp61F expression.

Overall, the various changes in signaling are quite puzzling-while Jak/Stat signaling from the niche is reduced, hh signaling appears to be increased. Similarly, while the authors conclude that premature differentiation occurs close to the niche, EGF signaling, which occurs from germ cells to cyst cells during differentiation, is decreased. Many times these changes are contradictory, and the authors do not provide a suitable explanation to resolve these contradictions.

Reviewer #2 (Public Review):

Summary:

In this work, the authors investigate the role of the Superoxide disumutase 1 (Sod1) enzyme, which acts to reduce the reactive oxygen species load, in the Drosophila testis. They show that the knockdown of Sod1 in somatic cells impacts stem cell numbers both autonomously in the soma and non-autonomously in the germline. Somatic stem cell numbers are increased, while germline stem cells are decreased and differentiate prematurely. The authors then show that in somatic Sod1 knockdowns, several signalling pathways are disrupted and that these may be responsible at least in part for the phenotypes observed. Finally, over-expression of Sod1 in the soma results in opposite phenotypes, suggesting that ROS levels do play a role in maintaining the balance between both stem cell populations in the testis.

Strengths:

The main strength of this work is to show a previously unappreciated role for Sod1 (and presumably by extension of ROS) in the Drosophila testis and in the regulation of stem cell self-renewal and differentiation. The authors use multiple readouts to show that the knockdown of Sod1 in the soma increases the number of somatic cells and also drives a non-autonomous, premature differentiation of germ cells. They also quantify the early differentiation of the germline using two different methods. Importantly, overexpression of Sod1 produces opposite phenotypes to knockdown, strengthening the conclusions.

Weaknesses:

Although the data presented are interesting, an important weakness of the manuscript as it currently stands is that many statements are not fully supported by the data. In particular, the authors do not provide any evidence of "cell redox-pairs" as indicated in the manuscript title, nor of intercellular redox gradients, as stated in several places throughout. While the data are consistent with non-autonomous regulation of ROS levels, this would not constitute a gradient. However, and crucially, there is no evidence provided to show that Sod1 manipulation in the soma is affecting ROS levels in the germline and that any of the phenotypes observed are a consequence of elevated ROS in the germline, rather than indirect effects caused by dysregulation of somatic self-renewal and differentiation, which is known to impact the germline. Indeed, there are many published reports of autonomous manipulations in the soma that influence either germline stem cell number (eg PMID: 19797664 among others) or differentiation (eg PMID: 17629483). The latter example is particularly relevant as the authors show altered somatic ERK levels, and the role of somatic ERK in promoting germ cell development is well established (PMID: 11048722, 11048723,...). Thus, whether Sod1 plays any non-autonomous role in controlling germ cell fate through ROS in the germline directly, or whether the phenotypes observed can all be explained by autonomous effects on somatic cell behaviour is debatable, but the experiments presented here do not distinguish between these two hypotheses. The only evidence presented by the authors for a non-autonomous role of Sod1 is the expression of a GFP reporter for gstD1. The quantifications and images are not clear and do not show unambiguously that this reporter is expressed in germ cells. Indeed, the quantifications show overlap between somatic and germline markers, suggesting that either the images themselves or the way they are quantified does not allow the authors to distinguish between the two cell types. Similarly, the claim that somatic mitochondria are enriched at the CySC-GSC interface and that this distribution maintains the redox balance in the niche is not supported by any experimental data. CySCs are extremely thin cells and much of the space is occupied by the nucleus (PMID: 114676), therefore it is likely that mitochondria would be enriched at the periphery. A careful analysis would be necessary to show that this enrichment is specific to the interface with GSCs. Moreover, no experiments are conducted to test whether mitochondrial distribution in CySCs has any impact on GSCs. Finally, no experiments are conducted to show definitively that the phenotypes observed upon Sod1 knockdown are indeed due to increased ROS, while this claim is made several times in the text. At present, the data presented here can support a role for Sod1 in somatic CySCs, but much more caution is required in attributing this to either ROS or intercellular ROS signaling. Therefore, several claims made in the title and throughout the text are not supported by evidence.

Besides this central point, there are other areas that should be improved. In particular, the data using the Fucci reporter to show accelerated proliferation do not appear convincing. It would seem that the proportions of cells in each phase are roughly similar, just that there are more cycling cells. A careful analysis of these results would distinguish between these two and determine whether Sod1 knockdown simply impairs differentiation (and therefore results in more somatic cells proliferating) or whether it speeds up the cell cycle (resulting in an increased mitotic index as suggested, but this requires a ratio to be shown). Similarly, several quantifications are not clearly explained, making it hard to understand what is being measured. As an example, while the decrease in pERK in CySCs is clear from the image and matched in the quantification, the increase in cyst cells is not apparent from the fire LUT used. The change in fluorescence intensity therefore may be that more cells have active ERK, rather than an increase per cell (similar arguments apply to the quantifications for p4E-BP or Ptc). Therefore, it is hard to know whether Sod1 knockdown results in increased or decreased signaling in individual cells.

Impact of study:

Demonstrating intercellular communication through ROS and its importance in maintaining the balance between two stem cell populations would be a finding of interest to a broad field. However, it remains to be demonstrated that this is the case, and given this, this study will have a limited impact.

Reviewer #3 (Public Review):

Summary:

The authors want to prove that there is a redox potential between germline stem cells (GSCs) and somatic cyst stem cells (CySCs) in the Drosophila testis, with ROS being higher in the former compared to the latter. They also want to prove that ROS travels from CySCs to GSCs. Finally, they begin to characterize the phenotypes caused by loss of SOD (which normally lowers ROS levels) in the tj- lineage and how this impacts the germline.

Strengths:

The role of SOD in somatic support cells is an under-explored area.

Weaknesses:

The authors fall short of accomplishing their goals. There are issues with the concept of the paper (ROS gradient between cells that causes a transfer of ROS across membranes for homeostasis), the data, the figures, and the scholarship of the testis. I have discussed each of the points in detail below. These weaknesses negatively impact the conclusions put forward by the authors. In short, their data is not compelling: there is no evidence provided by the authors that ROS diffuses from CySCs to GSCs as most of the claims about stem cells are founded on data about differentiating germ and somatic cells. The somatic SOD depletion phenotype is incompletely characterized and several pathways appear to change in these cells, including reduced Egfr signaling, increased Tor signaling, and increased Hh signaling. None of these results are sufficiently followed up on. And none of them are considered relative to their known roles in the testis. For example, high Hh signaling in CySCs increases their competitiveness with GSCs. Increased Tor signaling in all CySCs does not affect the CySC lineage. Reduced Egfr signaling in CySCs reduces the number of CySCs and reduces/inhibits abscission between GSCs-gonialblasts.

Major issues:

(1) Data
a. Problems proving which mitochondria are associated with which lineage.
b. There is no evidence that ROS diffuses from CySCs into GSCs.
c. The changes in gst-GFP (redox readout) are possibly seen in differentiating germ cells (i.e., spermatogonia) but not in GSCs. This weakens their model that ROS in CySC is transferred to GSCs.
d. Most of the paper examines the effect of SOD depletion (which should increase ROS) on the CySC lineage and GSC lineage. One big caveat is that tj-Gal4 is expressed in hub cells (Fairchild, 2016) so the loss of SOD from hub cells may also contribute to the phenotype. In fact, the niche in Figure 2D looks larger than the niche in the control in Figure 2C, arguing that the expression of Tj in niche cells may be contributing to the phenotype. The authors need to better characterize the niche in tj>SOD-RNAi testes.
e. The tj>SOD-RNAi phenotype is an expansion of the Zfh1+ CySC pool, expansion of the Tj+ Zfh1- cyst cells (both due to increased somatic proliferation) and a non-autonomous disruption of the germline.
f. I am not convinced that MAPK signaling is decreased in tj>SOD-i testes. Not only is this antibody finicky, but the authors don't have any follow-up experiments to see if they can restore SOD-depleted CySCs by expressing an Egfr gain of function. Additionally, reduced Egfr activity causes fewer somatic cells (not more) (Amoyel, 2016) and also inhibits abscission between GSCs and gonialblasts (Lenhart 2015), which causes interconnected cysts of 8- to 16 germ cells with one GSC emanating from the hub.
g. The increase in Hh signaling in SOD-depleted CySCs would increase their competitiveness against GSCs and GSCs would be lost (Amoyel 2014). The authors need to validate that Hh protein expression is indeed increased in SOD-depleted CySCs/cyst cells and which cells are producing this Hh. Normally, only hub cells produce Hh (Michel, 2012; Amoyel 2013) to promote self-renewal in CySCs.
h. The increase in p4E-BP is an indication that Tor signaling is increased, but an increase in Tor in the CySC lineage does not significantly affect the number of CySCs or cyst cells (Chen, 2021). So again I am not sure how increased Tor factors into their phenotype.
i. The over-expression of SOD in CySCs part is incomplete. The authors would need to monitor ROS in these testes. They would also need to examine with tj>SOD affects the size of the hub.

(2) Concept
Why would it be important to have a redox gradient across adjacent cells? The authors mention that ROS can be passed between cells, but it would be helpful for them to provide more details about where this has been documented to occur and what biological functions ROS transfer regulates.

(3) Issues with scholarship of the testis
a. Line 82 - There is no mention of BMPs, which are the only GSC-self-renewal signal. Upd/Jak/STAT is required for adhesion of GSCs to the niche but not self-renewal (Leatherman and Dinardo, 2008, 2010). The author should read a review about the testis. I suggest Greenspan et al 2015. The scholarship of the testis should be improved.
b. Line 82-84 - BMPs are produced by both hub cells and CySCs. BMP signaling in GSCs represses bam. So it is not technically correct to say the CySCs repress bam expression in GSCs.
c. Throughout the figures the authors score Vasa+ cells for GSCs. This is technically not correct. What they are counting is single, Vasa+ cells in contact with the niche. All graphs should be updated with the label "GSCs" on the Y-axis.

(4) Issues with the text
a. Line 1: multi-lineage is not correct. Multi-lineage refers to stem cells that produce multiple types of daughter cells. GSCs produce only one type of offspring and CySCs produce only one type of offspring. So both are uni-lineage. Please change accordingly.
b. Lines 62-75 - Intestinal stem cells have constitutively high ROS (Jaspar lab paper) so low ROS in stem cell cells is not an absolute.
c. Line 79: The term cystic is not used in the Drosophila testis. There are cyst stem cells (CySCs) that produce cyst cells. Please revise.
d. Line 90 - perfectly balanced is an overstatement and should be toned down.
e. Line 98 - division of labour is not supported by the data and should be rephrased.
f. Line 200 - the authors provide no data on BMPs - the GSC self-renewal cue - so they should avoid discussing an absence of self-renewal cues.

(5) Issues with the figures
a. The images are too small to appreciate the location of mitochrondria in GSCs and CySCs.
b. Figure 1
i. cell membranes are not marked, reducing the precision of assigning mitochondria to GSC or CySCs. It would be very helpful if the authors depleted ATP5A from GSCs and showed that the puncta are reduced in these cells and did a similar set of experiments for the tj-Gal4 lineage. It would also be very helpful if the authors expressed membrane markers (like myr-GFP) in the GSC and then in the CySC lineage and then stained with ATP5A. This would pinpoint in which cells ATP5A immunoreactivity is occurring.
ii. The presumed changes in gst-GFP (redox readout) are possibly seen in differentiating germ cells (i.e., spermatogonia) but not in GSC.
iii. Panels F, Q, and S are not explained and currently are irrelevant.
c. Figure 3K - The evidence to support less Ecad in GSCs in tj>SOD-i testes is not compelling as the figure is too small and the insets show changes in Ecad in somatic cells, not GSC.
d. Figure 4:
i. Panel A, B The apparent decline (not quantified) may not contribute to the phenotype.
ii. dpERK is a finicky antibody and the authors are showing a single example of each genotype. This is an important experiment because the authors are going to use it to conclude that MAPK is decreased in the tj>SOD-i samples. However, the authors don't have any positive (dominant-active Egfr) or negative (tj>mapk-i). As is standing the data are not compelling. The graph in F does not convey any useful information.
e. Figure S1D - cannot discern green on black. It is critical for the authors to show monochromes (gray scale) for the readouts that they want to emphasize. I cannot see the green on black in Figure S1D.
f. Figure S4 - there is no quantification of the number of Tj cells in K-N.

(6) Issues with Methods
a. Materials and Methods are not described in sufficient depth - please revise.
b. Note that tj-Gal4 has real-time expression in hub cells and this is not considered by the authors. The ideal genotype for targeting CySCs is tjGal4, Gal80TS, hh-Gal80. Additionally, the authors do not mention whether they are depleting throughout development into adulthood or only in adults. If the latter, then they must have used a temperature shift like growing the flies at 18C and then upshifting to 25C or 29C during adult stages.
c. The authors need to show data points in all of the graphs. Some graphs do this but others do not.
d. The authors state that all data points are from three biological replicates. This is not sufficient for GSC and CySC counts. Most labs count GSCs and CySCs from at least 10 testes of the correct genotype.